The primary focus of the section is to further our understanding of the molecular basis of signaling between G protein coupled receptors and voltage gated ion channels in neurons using electrophysiological, molecular, and imaging techniques. A recently completed project provides the first detailed description of gene structure and sequence for the human gene FFAR3 (GPR41) and an adjacent duplicated gene, GPR42. FFAR3 is a G-protein coupled receptor for which short-chain fatty acids (acetate, propionate, and butyrate) serve as endogenous ligands. The receptor is found on gut enteroendocrine L-cells, pancreatic &#946;-cells, and sympathetic neurons and has been implicated in a number of conditions including obesity, diabetes, allergic airway disease, and altered immune function. In primates, FFAR3 is part of a tandem segmental duplication that results in a duplicon termed GPR42 that shares > 99% sequence identity with FFAR3. The high sequence identity renders short-read sequencing of FFAR3 and GPR42 unreliable. Moreover, GPR42 is classified as a suspected pseudogene based on lack of function when heterologously expressed. In this study, we sequenced FFAR3 and GPR42 open reading frames from 56 individuals and found an unexpectedly high frequency of polymorphisms contributing to several complex haplotypes. We also identified a frequent (20%) structural variation that results in GPR42 copy number polymorphism. Finally, sequencing revealed that 50% of GPR42 haplotypes differed from FFAR3 by only a single non-synonymous substitution and that the GPR42 reference sequence matched only 4.4% of the alleles. Sequencing of cDNA from human sympathetic ganglia and colon revealed processed transcripts matching the individual's GPR42 genotype. Expression of several GPR42 haplotypes in rat sympathetic neurons uncovered a variety of pharmacological phenotypes that differed in potency and efficacy. Our data suggest that GPR42 be reclassified as a functioning gene and that recognition of sequence and copy number polymorphism of the FFAR3/GPR42 complex be considered during genetic and pharmacological investigation of these receptors. Puhl HL III, Won Y-J, Lu VB, Van B Lu, Ikeda SR. 2015. Human GPR42 is a transcribed multisite variant that exhibits copy number polymorphism and is functional when heterologously expressed. Sci Rep 5: 12880. A second completed project described the expression pattern of voltage-gated sodium channel, Nav1.8 (encoded by the Scn10a gene). Under physiological conditions, the voltage-gated sodium channel Nav1.8 is expressed almost exclusively in primary sensory neurons. The mechanism restricting Nav1.8 expression is not entirely clear, but we have previously described a 3.7 kb fragment of the Scn10a promoter capable of recapitulating the tissue-specific expression of Nav1.8 in transfected neurons and cell lines (Puhl and Ikeda, 2008). To validate these studies in vivo, a transgenic mouse encoding EGFP under the control of this putative sensory neuron specific promoter was generated and characterized in this study. Approximately 45% of dorsal root ganglion neurons of transgenic mice were EGFP- positive (mean diameter = 26.5 um). The majority of EGFP-positive neurons bound isolectin B4, although a small percentage (approximately 10%) colabeled with markers of A-fiber neurons. EGFP expression correlated well with the presence of Nav1.8 transcript (95%), Nav1.8- immunoreactivity (70%), and TTX-R INa (100%), although not all Nav1.8-expressing neurons expressed EGFP. Several cranial sensory ganglia originating from neurogenic placodes, such as the nodose ganglion, failed to express EGFP, suggesting that additional regulatory elements dictate Scn10a expression in placodal-derived sensory neurons. EGFP was also detected in discrete brain regions of transgenic mice. Quantitative PCR and Nav1.8-immunoreactivity confirmed Nav1.8 expression in the amygdala, brainstem, globus pallidus, lateral and paraventricular hypothalamus, and olfactory tubercle. TTX-R INa recorded from EGFP-positive hypothalamic neurons demonstrate the usefulness of this transgenic line to study novel roles of Nav1.8 beyond sensory neurons. Overall, Scn10a-EGFP transgenic mice recapitulate the majority of the Nav1.8 expression pattern in neural crest-derived sensory neurons. Lu VB, Ikeda SR, Puhl HL. 2015. A 3.7 kb fragment of the mouse SCN10a gene promoter directs neural crest but not placodal lineage EGFP expression in a transgenic animal. J Neurosci 35: 80218034. In addition to these projects, two collaborative manuscripts were published: 1) Iyer MR, Cinar R, Liu J, Godlewski G, Szanda G, Puhl H, Ikeda SR, Deschamps J, Lee Y-S, Steinbach PJ, et al. 2015. Structural basis of species-dependent differential affinity of 6-alkoxy-5-aryl-3-pyridinecarboxamide cannabinoid-1 receptor antagonists. Mol Pharmacol 88: 238244. We provided cannabinoid receptor mutants used in this study which exposed pharmacological differences between rodent (rat and mouse) and human receptors. 2) Nguyen TA, Sarkar P, Veetil JV, Davis KA, Puhl HL, Vogel SS. 2015. Covert changes in CAMKII holoenzyme structure identified for activation and subsequent interactions. Biophys J 108: 21582170. Dr. Henry Puhl provided training and advice for generating CamKII constructs used in this study of enzyme structure function using optical techniques.